Inducible Costimulator Is Essential for Collagen-Induced Arthritis

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Amendment history: Retraction (November 2003) Inducible costimulator is essential for collagen-induced arthritis

Roza I. Nurieva, … , Richard A. Flavell, Chen Dong

J Clin Invest. 2003;111(5):701-706. https://doi.org/10.1172/JCI17321.

Article Autoimmunity

CD4+ helper Th cells play a major role in the pathogenesis of rheumatoid arthritis. Th cell activation, differentiation, and immune function are regulated by costimulatory molecules. Inducible costimulator (ICOS) is a novel costimulatory receptor expressed on activated T cells. We, as well as others, recently demonstrated its importance in Th2 cytokine expression and Ab class switching by B cells. In this study, we examined the role of ICOS in rheumatoid arthritis using a collagen-induced arthritis model. We found that ICOS knockout mice on the DBA/1 background were completely resistant to collagen-induced arthritis and exhibited absence of joint tissue inflammation. These mice, when immunized with collagen, exhibited reduced anti-collagen IgM Ab’s in the initial stage and IgG2a Ab’s at the effector phase of collagen- induced arthritis. Furthermore, ICOS regulates the in vitro and in vivo expression of IL-17, a proinflammatory cytokine implicated in rheumatoid arthritis. These data indicate that ICOS is essential for collagen-induced arthritis and may suggest novel means for treating patients with rheumatoid arthritis.

Find the latest version: https://jci.me/17321/pdf Inducible costimulator is essential for collagen-induced arthritis

Roza I. Nurieva,1 Piper Treuting,2 Julie Duong,1 Richard A. Flavell,3 and Chen Dong1

1Department of Immunology, and 2Department of Comparative Medicine, University of Washington School of Medicine, Seattle, Washington, USA 3Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut, USA

CD4+ helper Th cells play a major role in the pathogenesis of rheumatoid arthritis. Th cell activation, differentiation, and immune function are regulated by costimulatory molecules. Inducible costimulator (ICOS) is a novel costimulatory receptor expressed on activated T cells. We, as well as others, recently demonstrated its importance in Th2 cytokine expression and Ab class switching by B cells. In this study, we examined the role of ICOS in rheumatoid arthritis using a collagen-induced arthritis model. We found that ICOS knockout mice on the DBA/1 background were completely resistant to collagen-induced arthritis and exhibited absence of joint tissue inflammation. These mice, when immunized with collagen, exhibited reduced anti-collagen IgM Ab’s in the initial stage and IgG2a Ab’s at the effector phase of collagen-induced arthritis. Furthermore, ICOS regulates the in vitro and in vivo expression of IL-17, a proinflammatory cytokine implicated in rheumatoid arthritis. These data indicate that ICOS is essential for collagen-induced arthritis and may suggest novel means for treating patients with rheumatoid arthritis. J. Clin. Invest. 111:701–706 (2003). doi:10.1172/JCI200317321.

Introduction haps by producing proinflammatory cytokines, cause Rheumatoid arthritis (RA) is a common autoimmune inflammatory reactions that involve macrophages, disease characterized by autoantibody-mediated neutrophils, and mast cells (7). T cell cytokines such as chronic inflammation of multiple joints and destruc- IL-17 have been found in the synovium of arthritis tion of cartilage and bone (1). Collagen-induced patients or CIA mice (8, 9), although the local regula- arthritis (CIA) is the most widely used mouse model tion of their expression has not been understood. for RA (2, 3), in which immunization of genetically Th cell activation, differentiation, and immune susceptible strains of mice, such as DBA/1, with type function are regulated by costimulatory molecules. II collagen (CII) leads to the development of a severe CD28 is the most important costimulatory receptor polyarticular arthritis mediated by immune mecha- on T cells. It binds to B7.1 (CD80) and B7.2 (CD86) on nisms. The autoimmune response in the CIA animal activated APCs. Studies using T cells derived from model involves CII-specific T cells and B cells and their CD28-deficient mice demonstrated its role in T cell products, proinflammatory cytokines, and anti-CII activation (10). Consistent with this, CD28 knockout Ab’s. There are considerable data to implicate CII-reac- mice on the DBA/1 background are resistant to CIA tive CD4+ cells as primary mediators of disease induc- (11). The function of CD28, however, in the effector tion and autoantibody production by B cells as a phase is less dominant. This is probably due to the major immune mechanism leading to the localized induction of its antagonist, CTLA-4, on activated T chronic inflammatory response (4–6). In addition to cells, which binds the same ligands with much higher providing helper cytokines such as IFN-γ to mediate affinity and functions to inhibit T cell proliferation Ab production by B cells, T cells but not B cells are and cytokine expression (12). often found to infiltrate into the joint tissues and, per- Inducible costimulator (ICOS) is the third member of the CD28 family. Not expressed by naive Th cells, it is induced after T cell activation (13, 14). The ligand for Received for publication November 5, 2002, and accepted in revised form January 7, 2003. ICOS, B7H (also named B7RP-1) is constitutively expressed on B cells and in nonlymphoid tissues such Address correspondence to: Chen Dong, Department of as the lung (13, 15). Most interestingly, B7H expression Immunology, University of Washington, Box 357650, α H466 Health Science Complex, Seattle, Washington 98195-7650, is strongly induced in fibroblasts by TNF- and in sev- USA. Phone: (206) 221-6694; Fax: (206) 543-1013; eral nonlymphoid tissues after LPS injection in mice E-mail: [email protected]. (15). Recently, we, as well as others, constructed and Conflict of interest: The authors have declared that no conflict of analyzed mice deficient in the ICOS gene (16–18). interest exists. ICOS-deficient mice exhibited impaired Ig class-switch- Nonstandard abbreviations used: rheumatoid arthritis (RA); collagen-induced arthritis (CIA); chicken type II collagen (CII); ing and germinal center reactions (16, 17). On the other –/– inducible costimulator (ICOS); prostaglandin E2 (PGE2). hand, we found that ICOS mice were extremely sen-

The Journal of Clinical Investigation | March 2003 | Volume 111 | Number 5 701 sitive to experimental autoimmune encephalomyelitis (EAE) (16). Therefore, ICOS appears to play opposite roles in humoral and inflammatory responses. The function of ICOS in Ab-mediated autoimmune dis- eases such as RA has not been addressed, however. In this paper, we have analyzed the role of ICOS in RA using the CIA model. We found that ICOS knockout mice on the DBA/1 background were completely resistant to CIA. Compared with ICOS+/+ mice, ICOS–/– exhibited reduced anti-collagen IgM and IgG2a titers, indicating that ICOS regulates to some extent the humoral responses. Furthermore, ICOS regulates the expression of IL-17 in vitro and in vivo. Therefore, ICOS plays an important role in the pathogenesis of CIA; it therefore provides an important and novel tar- get for therapy of RA patients.

Methods Mice. ICOS knockout mice were backcrossed six gener- ations onto the DBA/1 background. ICOS+/+ and –/– ICOS mice, all on F6 backcross, were interbred, and their offspring were used for the experiments. All mice were 8–10 weeks old at the time of immunization. The mice were housed in a specific pathogen-free facility. Induction of collagen-induced arthritis. Arthritis was induced and scored in male ICOS+/+ and ICOS–/– mice by immunizing with CII (Sigma-Aldrich, St. Louis, Mis- souri, USA) (19). Chicken CII was dissolved in cold 10 mM acetic acid overnight at 4°C and emulsified with equal volume of CFA (Sigma-Aldrich) to give a final Figure 1 Resistance to collagen-induced arthritis by ICOS-deficient mice. concentration 1 mg/ml. Experimental mice were ICOS–/– and ICOS+/+ mice on DBA/1 background were immunized at immunized intradermally at the base of the tail with the base of the tail on days 0, 21, and 42 with type II chicken colla- 100 µl of emulsified chicken CII on day 0 and boosted gen in CFA. (a) Disease severity was scored on days 38, 42, 47, 48, with the same agent on day 21 and 42. Severity of dis- 50, 55, and 58 by visual inspection of mouse paws. Each paw was ease was evaluated by visual inspection of the paws. scored for the degree of inflammation on a scale 0–4 as indicated in Each paw was scored for the degree of inflammation on Methods. Scores were then added together. The data shown are a a scale from 0 to 4: 0, no evidence of erythema and representative of two experiments on a total of more than ten mice swelling; 1, erythema and mild swelling confined to the in each group, with similar results. (b) Representative photos of forepaws and hindpaws of ICOS+/+ and ICOS–/– mice 58 days after midfoot (tarsals) or ankle joint; 2, erythema and mild first immunization with chicken CII. swelling extending from ankle to the midfoot; 3, erythema and mild swelling extending from ankle to metatarsal joints; 4, erythema and severe swelling µg/ml chicken CII. Collagen-specific Ab’s were detect- encompassing the ankle, foot, and digits. Scores from ed with biotinylated goat anti-mouse IgM and rat anti- all four paws were added to give the total for each ani- mouse IgG1 and IgG2a Ab’s (Southern Biotechnology mal. The experiments were performed in accordance Associates, Birmingham, Alabama, USA). with the regulation of the Animal Care Committee of T cell cytokine measurement. Spleen cells from ICOS+/+ University of Washington. and ICOS–/– mice 14 days after the first immunization Histopathological analyses. Experimental mice were euth- with chicken CII were either unstimulated or stimulat- anized by CO2 inhalation. Routine necropsy was per- ed with 50 or 100 µg chicken CII, and cytokine pro- formed, and the distal appendages were placed in 10% duction (IFN-γ, TNF-α, IL-4, and IL-17) in the 3-day neutral-buffered formalin for 7 days. The tissue sections culture supernatant was determined by ELISA were processed for routine paraffin embedding, and (PharMingen, San Diego, California, USA). Prolifera- hematoxylin and eosin staining following decalcification. tion was assayed after 3 days of treatment with differ- Measurement of anti-CII Ab’s. Anti-collagen Ab (IgM, ent doses of chicken CII by adding 3H-thymidine to the IgG1, IgG2a) production was measured in the serum of culture for the last 8 hours. To further test the role of mice either 14 days after the first immunization or at ICOS in costimulation of DBA/1 T cells, naive CD4+ T the end of the experiment (day 58) by ELISA. Briefly, cells from lymph nodes and spleens of 6- to 8- week-old serum samples from immunized mice were added in a wild-type DBA/1 mice were isolated, as we reported pre- threefold serial dilution on plates precoated with 10 viously (16). Purified naive CD4+ T cells were stimulat-

702 The Journal of Clinical Investigation | March 2003 | Volume 111 | Number 5 Figure 2 Representative hematoxylin and eosin–stained sections of intrapha- langeal joints from ICOS+/+ and ICOS–/– mice. (a) ICOS+/+. Notice the even and clear joint space (js) and smooth articular cartilage (ac). Original magnification ×200. (b–f) ICOS–/–. (b) Notice the fibrovascular synovial and periarticular proliferation (fp) erosion of articular cartilage (ac) intra-articular exudates (ie) and osteolysis in severely affected joints (d). Original magnification ×200. (c) Origi- nal magnification ×100. (d) Articular bone remnants (br) from a severely affected mouse. Original magnification ×100. (e) Intra- articular exudates composed of neutrophils (n), other inflammatory cells, and hyperplastic synoviocytes (s) admixed within cellular debris. Original magnification ×400. (f) High magnification of the fibrovascular proliferative tissues composed of primarily hyperplastic fibrocytes (hf), neovascularization (v), and inflammatory cells (ic). Original magnification ×600.

tail on day 0, 21, and 42 with CII in CFA. ICOS+/+ mice developed severe joint inflammation as seen by others, evidenced by marked swelling and erythema of the hindpaws and forepaws (Figure 1a). Inflammation in these paws included the wrist and ankle and extended distally through the limb and digits (Figure 1b). In con- trast, ICOS–/– DBA/1 mice were uniformly resistant to CIA (Figure 1a) and had no sign of paw or joint ed in triplicate with plate-bound anti-CD3 in the swelling (Figure 1b). absence or presence of anti-CD28 and/or anti-ICOS. We further examined the experimental mice histo- On day 4, Th cytokine production in the supernatant logically. Only the ICOS+/+ DBA/1 mice with clinical was measured by ELISA (PharMingen). To examine disease had histopathological lesions consistent with CD40L expression, lymph node cells from ICOS+/+ and collagen-induced arthritis. We examined the pedal ICOS–/– mice were activated with αCD3 (3 µg/ml) and joints of ICOS+/+ DBA/1 mice and ICOS–/– DBA/1 mice then stained with a biotinylated αCD40L Ab, followed (Figure 2). All of the ICOS–/– DBA/1 joints were histo- by streptavidin–R-phycoerythrin (Southern Biotech- logically normal with no sign of tissue degeneration nology Associates) for flow-cytometry analysis. and inflammation. All of the ICOS +/+ DBA/1 mice had severe histopathological lesions characterized by exten- Results sive fibrovascular and proliferative synovitis composed RA is an autoimmune disease affecting a large percent- of abundant fibroblasts, hypertrophic synoviocytes, age of the human population. CD4+ T cells play crucial and marked infiltration of inflammatory cells, prima- roles in the pathogenesis of the disease. How their rily neutrophils and macrophages, lesser lymphocytes, function is regulated, however, is still poorly under- and mast cells (Figure 2, b–f), which extended into the stood. In the current study, we examined the role of joint space. Additionally, in severely affected joints, ICOS costimulatory receptor in CIA. there was moderate to severe cartilage destruction, and ICOS knockout mice are completely resistant to CIA. We marked remodeling of bone. Often, the fibrovascular analyzed the role of ICOS in RA using the murine CIA model. We backcrossed ICOS knockout mice six gen- erations onto the DBA/1 background. ICOS–/– and ICOS+/+ DBA/1 mice were immunized at the base of the

Figure 3 Immunized ICOS–/– mice exhibited greatly reduced levels of anti-collagen IgM and IgG2a compared to ICOS+/+ mice. Anti-collagen Ab’s (IgM and IgG2a) were measured in the sera by ELISA. The sera from ICOS+/+ and ICOS–/– mice (three mice in each group), either 14 days after the first immunization or at the end of the experiment (day 58), were subjected to a threefold serial dilution; the concentrations of collagen-specific IgM and Ig2a were analyzed by ELISA, and average OD reading was indicated.

The Journal of Clinical Investigation | March 2003 | Volume 111 | Number 5 703 ed greatly reduced anti-CII IgM in serum (Figure 3). This result suggests an important role of ICOS in the initial B cell response, which can result in IgG2a deficiency and CIA resistance by ICOS–/– mice. T cell responses in the immunized ICOS–/– mice. We showed previously that ICOS is important for T cell activation and function (16). Defective B cell response in ICOS–/– could thus be caused by insufficient T cell priming or lack of certain T cell cytokine products. To analyze antigen-specific T cell responses in ICOS+/+ and ICOS–/– mice, we immunized ICOS+/+ and ICOS–/– with CII, and their spleen cells harvested 14 days later were restimulated with different doses of CII. T cell proliferation and cytokine secretion were examined. ICOS–/– cells exhibited moderately reduced T cell proliferation, suggestive of a minor role of ICOS in T cell priming in vivo (Figure 4a). Since the marginally reduced T cell priming described above could not fully explain the complete resistance of ICOS–/– mice to CIA, we further examined IFN-γ, TNF-α, IL-4, and IL-17 production by spleen cells in response to CII (Figure 4b). No IL-4 could be detected by either ICOS+/+ or ICOS–/– cells, correlating Figure 4 with the notion that the CIA protocol generates a Th1 Reduced proliferation and IL-17 expression by ICOS–/– T cells after response in vivo. No difference was observed in expres- CII immunization. ICOS+/+ and ICOS–/– mice were immunized with sion of IFN-γ in ICOS–/– and ICOS+/+ mice. Normal chicken CII in CFA. On day 14, spleen cells were isolated. (a) For the IFN-γ production by ICOS–/– mice would also argue µ proliferation assay, spleen cells were treated with 25, 50, 100 g/ml against a role of ICOS in regulating Ig class switching 3 chicken CII for 3 days. H-thymidine incorporation during the last 8 to IgG2a. In addition, TNF-α, an important cytokine hours was measured. (b) Cytokine production (IFN-γ, TNF-α, and µ for RA, was found to be produced normally in the IL-17) in supernatant of spleen cells activated with 50 or 100 g/ml α chicken CII for 3 days was measured by ELISA. absence of ICOS. Therefore, a TNF- –independent mechanism must confer CIA resistance in the knock-

proliferation and inflammation extended into the periarticular connective tissues and adjacent musculature. Reduced anti-CII humoral responses in the absence of ICOS. To better characterize the immune mechanisms underlying CIA resistance by ICOS–/– mice, we measured first anti-collagen Ab production (IgM, IgG1, IgG2a) in the serum of mice at the end of the experiments (day 58) (Figure 3). CIA is best characterized by pathogenic anti- CII IgG, especially IgG2a Ab’s. Consistent with the resistance of ICOS–/– mice to CIA, we found they exhibited greatly reduced levels of IgG2a Ab to anti-CII. On the other hand, low levels of anti-CII IgG1 production were not severely affected in these mice (data not shown). Interestingly, anti-CII IgM was also reduced in the absence of ICOS. ICOS has been shown previously by others and us to play an important role in regulating B cell Ig class switching (16–18, 20). We therefore examined only if Figure 5 class-switching was affected in the absence of ICOS. ICOS regulates IL-17 but not CD40L expression. (a) Naive Th cells Serum anti-CII Ab’s were measured from ICOS+/+ and from ICOS+/+ mice were stimulated with the indicated combination –/– of anti-CD3, anti-CD28, and anti-ICOS Ab’s. After 4 days, the ICOS mice 14 days after the first immunization. At γ this time point, there was little anti-collagen IgG1 and amounts of Th cytokines (IFN- and IL-17) were measured by ELISA. (b) Lymph node cells from ICOS+/+ and ICOS–/– mice were activated IgG2a production by both groups (data not shown). with αCD3 for 6 hours, stained with mAb to CD40L plus strepta- On the other hand, IgM was produced most signifi- vidin-phycoerythrin, and analyzed by flow cytometry (open curves). cantly by ICOS+/+ mice (Figure 3). Surprisingly, we Control staining is shown as dark curves. The data represent one of found at this stage that ICOS–/– DBA/1 already exhibit- two independent experiments with the same results.

704 The Journal of Clinical Investigation | March 2003 | Volume 111 | Number 5 out mice, suggesting that an anti-ICOS therapy for RA ICOS has been implicated previously in regulating may serve as an alternative for patients who fail CD40L expression by activated T cells (17). In addition TNF-α–targeted therapy. to T cell cytokines, we also examined the CD40L ICOS regulates IL-17 expression. Interestingly, spleen expression. ICOS+/+ and ICOS–/– lymph node cells were cells from ICOS–/– mice produced a remarkably activated with anti-CD3 for 6 hours, and cell-surface decreased amount of IL-17 in response to CII (Figure CD40L expression was examined by flow cytometry. 4b). IL-17 is a new cytokine secreted by CD4+-acti- Consistent with our previous report (16), ICOS–/– cells vated memory T cells and is frequently found in RA expressed CD40L at the same level as the ICOS+/+ cells. synovium (8, 21, 22). The cellular responses to IL-17 Therefore, it is unlikely that ICOS regulates CIA are similar to IL-1, including IL-6, IL-8, G-CSF, and response through a CD40L-dependent mechanism. –/– prostaglandin E2 (PGE2) production (8, 21). The In conclusion, we found ICOS mice on DBA/1 effect of IL-17 on cartilage was associated with its background to be completely resistant to CIA. ICOS–/– destruction, including activation of NO, and IL-17 mice exhibited reduced B cell response in anti-CII Ab has also been described as a potent stimulator of production. In addition, we report a novel role of osteoclastic bone reabsorption due to PGE2 synthe- ICOS in regulation of IL-17 expression. Since IL-17 sis (23–25). IL-17 produced by CD4+ cells in the joint has been shown to mediate joint tissue inflammation tissue may contribute to the inflammatory process and is important in humoral immune responses, involved in RA. In support of this notion, overex- ICOS thus possibly mediates autoimmune arthritis pression of IL-17 in the joints resulted in inflamma- by regulating IL-17 expression. Considering that tory infiltration of the synovium and aggressive car- ICOS is only expressed on activated T cells, anti-ICOS tilage degradation (26). Blockade of IL-17 action with approaches may therefore provide specific T cell– a soluble receptor significantly reduced the CIA, based therapy for RA patients. including a clear suppression of joint damage (27). Thus, defective IL-17 production in the ICOS–/– mice Acknowledgments may contribute to lack of joint inflammation in these We thank Charlene Karr-May for her histotechnologi- mice. Since ICOS ligand, B7H, is expressed in cal assistance, Durbaka V. R. Prasad for his assistance inflamed tissues, ICOS-B7H interaction may regulate in experimental procedures, and the entire Dong lab IL-17 local expression in the joint tissue in the CIA for their help and discussion. This work is supported model and mediate the inflammatory responses. On in part by a start-up fund from the Department of the other hand, a recent study of the IL-17 knockout Immunology at the University of Washington. Richard mice indicates a novel role for IL-17 in humoral Flavell is an investigator for the Howard Hughes Med- immune responses (28). Although the role of IL-17 in ical Institute, and Chen Dong is an Arthritis Investiga- autoantibody production in the CIA model has not tor award recipient from the Arthritis Foundation. been formally addressed, the reduced anti-CII auto- –/– 1. Feldmann, M., Brennan, F.M., and Maini, R.N. 1996. Rheumatoid arthri- antibodies in ICOS mice possibly could be attrib- tis. Cell. 85:307–310. uted to the IL-17 deficiency. 2. Luross, J.A., and Williams, N.A. 2001. The genetic and immunopatho- IL-17 has emerged as an important T cell–derived logical processes underlying collagen-induced arthritis. 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